ABSTRACT: The ?2 and ?4 alleles of APOE are by far the strongest and most well-established common genetic risk factors for Alzheimer disease (AD). The ?4/AD association varies with sex and age, whereas the protective effect of ?2 does not. The ?2/AD association is less well studied, in part because the ?2 allele is much less frequent than ?4. We will direct our efforts to the ?2/AD association for which we have recently obtained exciting preliminary results from analysis of datasets assembled by the Alzheimer Disease Genetics Consortium (ADGC). We identified genome-wide significant (GWS) association of AD with PPP2CB among ?2 carriers. PPP2CB encodes the catalytic subunit of protein phosphatase 2A, which is known to dephosphorylate tau protein through interaction with phosphatase methylesterase-1 (PME-1). This proposed project will fortify these and identify new associations using ADGC Haplotype Reference Consortium imputed GWAS datasets from multiple ancestries. We will identify functional variants in these genes by analysis of whole genome and whole exome data from the Alzheimer Disease Sequencing Project. We will also perform a variety of functional studies in brain tissue from pathologically confirmed AD cases and controls (with equal numbers of APOE ?2/?3, ?3/?3 and ?3/?4 genotypes) to delineate AD-related functional interactions of APOE ?2 mediated protective mechanisms involved in PP2A- tau pathway genes and other top genes from the GWAS. First, we will determine the effect of expression quantitative loci (eQTLs) and DNA methylation QTLs (mQTL of the ?2-interacting variants on AD-related traits. Next, we will evaluate associations between functional signatures of APOE ?2-interacting genes and protein levels of PPP2CB and neuroinflammatory genes, and quantitative neuropathological phenotypes. We will use the information from the genetic association study and brain experiments to understand how genetic variation leads to pathologic states as well as to provide models which can be used in small molecule drug screens for potential AD treatments. Specifically, we will establish in vitro proof-of-concept for differential effects of the interaction of APOE isoforms, PP2A and PME-1 on tau phosphorylation and accumulation using PME-1 inhibitors using the following approach. First, we will analyze pathogenic alteration of tau protein and PP2A activity in SH- SY5Y-Tau cells (APOE ?3/?3 genotype) in tissue cultured media containing dialyzed FBS and lipidated human APOE2 or APOE4 in the presence of PME-1 inhibitors or siRNA specific to PME-1. Next, using CRISPR, we will introduce a high-impact AD-associated PPP2CB variant to SH-SY5Y-Tau cells that have been cultured in the same manner to study its effect on pathogenic alteration of tau protein and PPP2CB activity. We will validate these findings in human iPS cells-derived neurons containing APOE ?2/?3 by CRISPR-mediated introduction of functional PPP2CB or PPME1 variants. These iPS cells will be analyzed for pathogenic changes of tau protein and PP2A activity. We expect that this project will identify methods to mimic the neuroprotective effect of ?2 which might lead to the development of effective drugs to treat or retard mechanisms leading to AD.